Lyme disease (Lyme borreliosis) is the most common tick-borne infectious disease in North America and Europe, and has been found in Russia, Japan, China and Australia. Lyme disease begins at the site of a tick bite, producing a primary infection with spread of the organism to secondary sites occurring during the course of infection. The causative bacterial agent of this disease is the spirochete Borrelia burgdorferi, which was first isolated and cultivated in 1982 (Burgdorferi, W. A. et al., Science 216: 1317-1319 (1982); Steere, A. R. et al., N. Engl. J. Med., 308:733-740 (1983)).
Three pathogenic genospecies of Borrelia, B. burgdorferi sensu stricto (B. burgdorferi or B.b.s.s.), B. afzelei and B. garinii have been described (Baranton, G., et al., Int. J. Syst. Bacteriol., 42:378-383 (1992)). These are members of a species complex, B. burgdorferi sensu lato, which consists of at least 10 different genospecies (Piken, R. N. et al., J. Invest. Dermatol., 110:211-214 (1998); Postic, D. et al., Int. J. Syst. Bacteriol., 44:743-752 (1994); Valsangiacomo, C. T. et al., Int. J. Syst. Bacteriol., 47:1-10 (1997)). The three genospecies, B. burgdorferi sensu stricto, B. afzelii and B. garinii, are all thought to be pathogenic and all are found in Europe.
B. burgdorferi has an outer membrane whose major protein constituents are the outer surface proteins A and B (OspA and OspB). OspA is a basic lipoprotein of approximately 31 kd, which is encoded on a large linear plasmid along with OspB, a basic lipoprotein of approximately 34 kd (Szczepanski, A., and J. L. Benach, Microbiol. Rev., 55:21 (1991)). The immune response to these outer surface proteins tends to occur late in the disease, if at all (Craft, J. E. et al., J. Clin Invest. 78:934-939 (1986); Dattwyler, R. J. and B. J. Luft, Rheum. Clin. North Am., 15:727-734 (1989)). Furthermore, patients acutely and chronically infected with B. burgdorferi respond variably to the different antigens, including OspA, OspB, OspC, OspD, p39, p41 and p93.
Currently, Lyme Disease is treated with a range of antibiotics, e.g., tetracyclines, penicillin and cephalosporins. However, such treatment is not always successful in clearing the infection. Treatment is often delayed due to improper diagnosis with the deleterious effect that the infection proceeds to a chronic condition, where treatment with antibiotics is often not useful. One of the factors contributing to delayed treatment is the lack of effective diagnostic tools.
Vaccines against Lyme borreliosis have been attempted. However, a vaccine that consists of recombinant OspA may require frequent booster immunizations. An additional concern of OspA-based vaccines is the recent identification of a putative autoreactive OspA domain with a high degree of similarity to a region of human leukocyte function-associated antigen-1 (hLFA-1) (Gross, D. M. et al., Science, 281: 703-706 (1998)).
Therefore, it should be advantageous to develop modified OspA proteins having decreased cross-reactivity to hLFA-1 in order to reduce potential side effects of an OspA vaccine. Development of OspA proteins with decreased hLFA-1 cross-reactivity that maintain or have increased immunoreactivity to more than one member of the Borrelia complex would also be desirable. To be useful as vaccines, the conformations of these modified proteins must be sufficiently stable to retain certain OspA structural features that are required to elicit a protective immune response. OspA proteins with these features would allow for improvements in diagnosis and/or vaccination against all, or most, of the Borrelia that cause Lyme Disease.
Analysis of the immune status of OspA immunized individuals revealed that the overall quantitative response is not predictive of protection, but rather the reactivity with a specific epitope of the OspA lipoprotein directly correlates to protective immunity. The anti-OspA monoclonal antibody, LA-2 (Kramer et al., 1990) defines an epitope of the lipoprotein that is apparently necessary for protective immunity after OspA vaccination. For instance, passive immunization of mice with this antibody leads to protection against infection with the spirochete (Schaible et al., 1993). In addition, immunization of mice and canines with OspA resulting in significant titers of LA-2 equivalent serum antibody accurately predicts protection from tick transmission of infection (Golde, 1997). Insufficient levels of LA-2 equivalent antibody result in a lack of protection in the face of high serum antibody titers to OspA (Johnson et al., 1995).